Neurotransmitters can effect cell function by binding to receptors associated with ion channels (ionotropic receptors) or to receptors that stimulate second messenger production (metabotropic receptors). The gating of ion channels results in changes in membrane potential that alters cell excitability. Second messengers often bring about an increase in intracellular Ca2+. Cytoplasmic Ca2+ transients trigger key cellular events including modification of excitability, cell growth and transmitter release. The long-term objective of the proposed research is to understand the mechanisms of action of neurotransmitters on ion permeation and Ca2+ metabolism in fish retinal horizontal cells. The specific aims are to determine the basic mechanisms of ion permeation initiated through ligand gated channels, the functional components of the proteins that constitute these channels, the changes in intracellular Ca2+ initiated by neurotransmitters that may be important in regulating the electrical activity of horizontal cells and the neurotoxic response. Two approaches will be used. One employs whole cell voltage clamp using patch electrodes to measure the membrane current produced by applied neurotransmitters and pharmacological agents that modify either the action of the transmitters or the amino acid composition of the receptor/channel protein. The second method will utilize the Ca2+ sensitive fluorescent indicator fura-2 to measure intracellular Ca2+ concentration in the presence of neurotransmitters. These experiments will be done on voltage clamped cells so as to distinguish the contributions to the changes in intracellular Ca2+ made by voltage sensitive Ca2+ channels and ligand gated channels. To carry out these studies, a concentration clamp system has been developed that permits the rapid exchange of solutions while maintaining the cell under voltage clamp with simultaneous measurement of cell fluorescence. Under these conditions, precise control of known concentrations of drugs is maintained. The measure of cell viability as a function of [Ca2+]i will be determined by cell shape and cell response to agonists or changes in membrane voltage. The results from this research will provide new and extensive information on many of the structural and biophysical properties of neurotransmitter- receptor interactions. The results of this investigation will have health- related implications since neurons rely on interactions with chemical reagents for several key functions.